DESCRIPTION (adapted from the application) The importance of iron is underscored by its participation in many cellular processes involving oxygen or redox reactions. Iron in excess of cellular needs is toxic; dietary overload or hereditary hemochromatosis leads to tissue iron deposition and injury, most likely due to redox activity of iron and consequent free radical reactions. The precise balance required to maintain appropriate cellular and tissue iron levels has led to mechanisms that regulate the synthesis of iron transport and storage proteins, e.g., transferrin receptor and ferritin. A role for copper in iron metabolism has been known for about 70 years. The important role for ceruloplasmin (Cp) in iron metabolism in vivo has been reinforced by the identification of "aceruloplasminemia" patients with Cp gene defects and massive iron deposits in many tissues, including the brain. This function for Cp has received support from studies showing that two Cp homologues, fet3p in yeast and hephaestin in mouse, play key roles in iron homeostasis. We have shown that the rate of Cp synthesis by HepG2 and Hep3B cells is tightly regulated by cellular iron status. Iron deficiency markedly increases Cp protein synthesis and gene expression. Nuclear "run-on" and mRNA stability studies indicate that regulation is by a transcriptional mechanism. We have new evidence that Cp transcription is regulated by hypoxia-inducible factor (HIF)-1 responsive elements since transcription is regulated by hypoxia and other HIF-1 activators. In addition, an enhancer element in the human Cp gene 5'-regulatory region contains HIF-1 responsive elements which increase reporter gene expression about 10-fold. In contrast to the stimulatory activity of iron deficiency, excess iron decreases Cp synthesis of HepG2 cells; surprisingly, a post-transcriptional mechanism has been observed in which Cp mRNA is destabilized by iron. These results support the important role of Cp in maintenance of cellular iron homeostasis. We hypothesize that the rate of hepatic Cp synthesis, like that of other proteins involved in iron transport, is regulated by cell iron status. In this application we will test the following specific aims: that a specific trans-activating factor(s) in iron-deficient HepG2 cells binds to a cis-acting element in the Cp 5'-regulatory region, thereby increasing Cp transcription. Furthermore, iron in excess alters the activity of a specific trans-acting factor(s) that binds to a region of the Cp 3'-UTR, thereby destabilizing the Cp mRNA. The long-term goal of this research is to understand the specific function of Cp in iron homeostasis, and especially its role in primary (genetic) and secondary (dietary) iron overload states.